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protein concentration and Pull down


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#1 NDUFB11

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Posted 04 January 2013 - 11:18 AM

Hello,
I have another question for you
I am going to set up a pull down assay using magnetic beads invitrogen.
In my previous experiment I performed a GST pull down experiment following a protocol inherited by my ex colleague.
Comparing this protocol, especially when I compared the concentration of proteins in the cell extract that people report on their papers, I noticed that I used a concentration of proteins which is too high ...sometime over then 6mg/ml of proteins whereas in the papers people report not more then 1-2 mg/ml.

My question is ..
Which is the criteria to use a correct amount of bait protein coupled as well as the protein concentration in the cell extract ?

thank you

-P

#2 Pangea

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Posted 04 January 2013 - 12:43 PM

Check the manual for the capacity of magnatic beads? If they are saturated its no matter how much you start with.

#3 NDUFB11

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Posted 04 January 2013 - 02:34 PM

So you mean I could use 10ug or 100ug of bait protein and obtain the same result (it sound a little bit weird for me though ) ,
and what about the cell extract ?
I don't think that in this case, using a concentration of protein either 1mg/ml or 8mg/ml the result is the same
it's true that there are a lot of variabilities like the affinity of my protein with the bait, if it's just one protein to pull down or more then one.
Probably the best way could be to make a test with different concentrations of cell extract protein

#4 Pangea

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Posted 05 January 2013 - 01:10 AM

No, of course if you use more magnetic beats you will increase you yield depending on your experiment design. But as I said you beads have capacity ana useing a lot of beats are not recommend. But in different concentration as you said,




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